INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
Toxicological evaluation of certain veterinary drugresidues in foodWHO FOOD ADDITIVES SERIES 39
Prepared by:
The forty-eighth meeting of the Joint FAO/WHO Expert
Committee on Food Additives (JECFA)
World Health Organization, Geneva 1997
FLUMEQUINE (addendum)
First draft prepared by
Professor F.R. Ungemach
Institute of Pharmacology, Pharmacy and Toxicology
Veterinary Faculty, University of Leipzig, Leipzig, Germany
1. Explanation
2. Biological data
2.1 Toxicological studies
2.1.1 Short-term toxicity
2.1.1.1 Arthropathy in dogs
2.1.1.2 Hepatotoxicity in mice
2.1.1.3 Mechanism of tumorigenicity in mice
2.1.2 Special studies on human intestinal flora
3. Comments
4. Evaluation
5. References
1. EXPLANATION
Flumequine is a fluoroquinolone antimicrobial agent. This
substance was evaluated by the Committee at its forty-second meeting
(Annex 1, reference 110). At that time, an ADI could not be
established owing to a lack of adequate information on the
toxicological and microbiological hazards of flumequine: (i) necessary
to identify a NOEL for hepatotoxicity; (ii) on the mechanism of
tumorigenesis; (iii) on the possible induction of arthropathy; and
(iv) on the microbiological safety of residues.
The additional information that was provided on these issues is
summarized in this monograph addendum.
2. BIOLOGICAL DATA
2.1. Toxicological data
2.1.1 Short-term toxicity
2.1.1.1 Arthropathy in dogs
Erosive arthropathy is a characteristic toxic reaction to
quinolones and has been observed in growing animals and in particular
young dogs after moderate doses of various fluoroquinolones (Gough
et al., 1979; Brown, 1996). Transient joint damage has also been
reported in children (Norrby & Lietman, 1993). An early three-month
study of the toxicity of flumequine in young adult beagle dogs did not
address arthropathic lesions of weight-bearing joints in particular
(Nelson et al., 1972).
Flumequine was administered twice daily as tablets by gavage to
groups of 10 three-month-old beagle dogs of each sex at doses of 0,
15, 30, 60, or 150 mg/kg bw per day for 13 consecutive weeks. Four
animals of each group were killed after three weeks. The absorption of
the test compound was checked periodically by high-performance liquid
chromatography analysis for flumequine and its 7-hydroxy metabolite in
plasma. The animals were observed daily for clinical signs, with
special attention to lameness and locomotor activity. Body weight was
recorded weekly. The serum activity of alkaline phosphatase was
measured on three occasions during the study. Twenty animals were
killed after three weeks of treatment, and the remainder were killed
at 13 weeks. All animals were submitted to necropsy and checked for
gross abnormalities and alterations of the articular surfaces of
weight-bearing joints of the fore- and hindlimbs; the shoulder and hip
joints were submitted to histopathological examination. The study was
conducted according to good laboratory practice (GLP) guidelines.
No deaths and only a few signs of adverse reactions were
observed, including vomiting and reduced food consumption which
increased in frequency with dose. No clinical signs of arthropathy,
such as staggering gait and reduced locomotor activity, were reported.
Females at the higher doses had markedly reduced weight gain. The
serum activity of alkaline phosphatase remained unchanged. Gross
necropsy revealed erosions of the joint surfaces in two of 10 dogs at
the highest dose and in one of 10 females at the lowest dose. Slight
histopathological lesions of the articular cartilage were observed in
one of 10 dogs given 60 mg/kg bw per day and three of 10 dogs given
150 mg/kg bw per day. These lesions were characterized by erosions,
cavities, a fibrillary appearance of the cartilage, and synovial
hyperplasia. The severity of the arthropathic lesions was similar at
three and 13 weeks. The gross lesions of the hip joints of one animal
after 13 weeks at the lowest dose were not accompanied by
histopathological alterations (Woehrle, 1996).
The grossly observed erosions in the hip joints of one female at
the lowest dose were considered to be spontaneous, since no related
histopathological alterations were found and no gross lesions were
observed at the next two higher doses. The NOEL for induction by
flumequine of arthropathy in young dogs was thus 30 mg/kg of bw per
day.
2.1.1.2 Hepatotoxicity in mice
In short-term and long-term studies of toxicity evaluated
previously by the Committee (Annex 1, reference 110), oral
administration of flumequine caused dose-related hepatotoxic effects
in rats and mice. Hypertrophy, degenerative changes, and focal
necrosis of hepatocytes were observed in rats at 800 mg/kg bw per day
in a three-month study (Nelson et al., 1972) and at 400 and 800
mg/kg bw per day in a two-year study (Sibinski et al., 1977a) and in
CD-1/ICR mice at 400 and 800 mg/kg bw per day in an 18-month study
(Sibinski et al., 1977b, 1979). The prevalence of hepatotoxic
lesions increased with the duration of treatment. After cessation of
flumequine administration, the liver damage was reversed (Sibinski
et al., 1979). Male mice were the most sensitive to
flumequine-induced liver damage (Sibinski et al., 1977b, 1979).
There was no NOEL for the hepatotoxic effects of flumequine in CD-1
mice.
In a 13-week study designed to investigate the hepatotoxic
lesions and the activity of hepatic drug-metabolizing enzymes,
flumequine was administered orally to groups of 16 CD-1 mice of the
same strain as used in the previous studies. The animals were fed
diets containing flumequine at concentrations providing doses of 0,
25, 50, 100, 400, or 800 mg/kg bw per day to males and 0, 100, 400, or
800 mg/kg bw per day to females. The concentration of the test
compound in the diet was checked periodically. The animals were
observed daily for clinical signs, and body weight, while food
consumption and food conversion efficiency were recorded weekly.
Plasma enzyme activities were measured once, after 12 weeks. On the
last day of treatment, the test compound was shown to be absorbed by
high-performance liquid chromatography analysis of flumequine and its
7-hydroxy metabolite in plasma. At the end of treatment, all animals
were necropsied, and complete macroscopic examinations were conducted.
The weights of the livers were recorded, and samples of the liver and
other grossly abnormal tissues were submitted to histopathology. At
the end of the experiment, liver microsomes were prepared to test the
activity of the xenobiotic-metabolizing enzyme system by measuring
total protein and cytochrome P450 content, P450-dependent dealkylation
of resorufin and coumarin derivatives, and 1-naphthol glucuronidation.
Microsomes from Aroclor-treated animals were used as positive
controls. The study was performed in compliance with GLP.
No deaths and no clinical signs of treatment-related adverse
reactions were observed. The body-weight gains of the animals at the
highest dose were reduced during the first week. This effect was more
pronounced in males, which also showed slightly lower food consumption
and efficiency of food conversion. No differences from the controls
were seen in the other groups during the study. Plasma enzyme
activities indicated liver damage at high doses, with a significant
increase in the activities of alanine aminotransferase and alkaline
phosphatase at doses of 400 and 800 mg/kg bw per day flumequine and of
lactic dehydrogenase and aspartate aminotransferase at 800 mg/kg bw
per day. Liver weights were increased at the two highest doses.
Histopathological examination of the livers revealed dose-dependent
degenerative alterations of hepatocytes, with hypertrophy and fatty
vacuolation (in males at doses greater than 25 mg/kg bw per day and in
females at doses greater than 100 mg/kg bw per day), increased ploidy,
intranuclear inclusions, and centrilobular necrosis (at doses greater
than 100 mg/kg bw per day). The effects were more pronounced in male
animals. Increased mitotic activity was observed in males at the
highest dose. Flumequine at doses up to 800 mg/kg bw per day had
little or no effect on P450-dependent hepatic drug-metabolizing
enzymes or on glucuronyltransferase activity.
It was concluded that (i) flumequine has no remarkable inducing
or inhibitory effect on the hepatic P450-dependent
xenobiotic-metabolizing enzyme system or on glucuronidation, and (ii)
the liver is the target organ of flumequine in mice. The degeneration
of hepatocytes with focal necrosis, accompanied by increased mitotic
activity indicating regenerative processes, was seen only in male mice
at the highest dose. The slight hypertrophic alterations of liver
cells with minimal degenerative alterations in males at doses of 50
and 100 mg/kg bw per day were regarded as signs of hepatotoxic lesions
rather than metabolic overload. The NOEL was thus 25 mg/kg bw per day
on the basis of hepatotoxic lesions in males (Stewart, 1995).
2.1.1.3 Mechanism of tumorigenicity in mice
The results of long-term studies with rodents previously
evaluated by the Committee (Annex 1, reference 110) showed no
carcinogenic effects in rats (Sibinski et al., 1977a), but a
dose-related increase in the incidence of benign and malignant liver
tumours was observed in CD-1 mice at doses greater than 100 mg/kg bw
per day. The tumour incidence was parallelled by hepatotoxic changes
and was significantly higher in male mice, which are known to be
sensitive to liver tumour induction (Sibinski et al., 1977b, 1979;
McClain, 1990). As the compound was inactive in a range of tests for
genotoxicity, including assays for gene mutation in bacteria and
mammalian cells in vitro and for chromosomal aberrations in
mammalian cells in vivo, the mechanism of tumorigenicity was
unclear.
The available toxicological database on flumequine and data from
the open literature were reviewed in order to discern the genotoxic or
non-genotoxic ('epigenetic') mechanism of the hepatocarcinogenicity of
flumequine (Marzin, 1996). By definition, a genotoxic carcinogen acts
directly on DNA in the target tissues, inducing DNA damage, strand
breaks, or mutations, which can be assessed in vitro in assays for
genotoxicity and in short-term assays in rodents. A non-genotoxic
carcinogen is devoid of such activity. The neoplastic response to a
non-mutagenic carcinogen is characterized by a steep dose-response
curve and a threshold dose. Non-genotoxic carcinogenicity is
considered to be brought about by potent induction of cytotoxicity and
cell proliferation, which may increase the frequency of tumours in the
target organs by virtue of sustained mitogenic stimuli. Demonstration
of a lack of genotoxic potential and of the induction of a
dose-related increase in cell proliferation thus remains critical for
identifying non-genotoxic carcinogens (Faccini et al., 1992;
Purchase, 1994; Shaw & Jones, 1994).
As negative results were obtained in various assays for
genotoxicity, including reverse mutation in Salmonella typhimurium,
gene mutation at the hprt locus in mouse lymphoma cells, gene mutation
in Chinese hamster cells, and chromosomal aberrations in bone-marrow
cells of rats in vivo, it is unlikely that flumequine or its
metabolites have direct genotoxic or mutagenic activity. Flumequine,
like other 4-quinolones, exerts its antibacterial effects at the level
of DNA by inhibiting bacterial topoisomerase II (DNA gyrase) (Hussy
et al., 1986; Sato et al., 1989). Although bacterial and
eukaryotic topoisomerases II share some structural homology, they
differ markedly in structure and function, which may explain the very
different sensitivities to the inhibitory activity of 4-quinolones
(Liu & Wang, 1991). Various 4-quinolones, including fluoroquinolones,
have an affinity for the mammalian enzyme that is several orders of
magnitude lower than that for the bacterial gyrase. Thus, the median
inhibitory dose (ID50) for calf thymus topoisomerase II exceeds the
ID50 for gyrase of Escherichia coli by at least 100 to 2460-fold
(in the case of loxacin) (Hussy et al., 1986; Sato et al., 1989).
This low affinity is assumed to be a common feature of all
fluoroquinolones. Although the inhibition of mammalian topoisomerase
II by flumequine was not investigated, it is unlikely to exist, even
at therapeutic doses. This conclusion is supported by the lack of
mutagenic potential of flumequine, whereas specific inhibitors of the
eukaryotic topoisomerase II are genotoxic and induce frameshift-type
mutations (Huff & Kreuzer, 1991).
Non-genotoxic tumorigenesis in the rodent liver can occur by
various mechanisms, including compound-related hormonal activity,
peroxisomal proliferation, induction of hepatic drug-metabolizing
enzymes, and hepatotoxicity.
The toxicological database, including the results of studies of
reproductive toxicity, revealed no evidence for any hormonal activity
of flumequine; and in short- and long-term assays for toxicity in
mice, no histopathological alterations of liver cells indicating
peroxisomal proliferation were reported.
Various inducers of the hepatic P450-dependent xenobiotic
biotransformation system, such as phenobarbital and halogenated cyclic
hydrocarbons, act as tumour promoters in rodent liver when
administered at high doses over a prolonged period (Diwan et al.,
1990; McClain, 1990; Grasso & Hinton, 1991). Since flumequine had only
negligible effects on the hepatic P450 enzyme system in the 13-week
study of toxicity in CD-1 mice at doses up to 800 mg/kg bw per day
(Stewart, 1995), induction of the hepatic xenobiotic-metabolizing
enzyme system can be excluded as a non-genotoxic mechanism of the
hepatocarcinogenicity of flumequine.
Flumequine is hepatotoxic, causing hepatocellular degeneration
and focal necrosis in male and female mice, followed by a mitogenic
response in male mice at the highest dose (Stewart, 1995). The
dose-related severity of these hepatotoxic lesions parallelled the
incidence of benign and malignant liver tumours (Sibinski et al.,
1977b). Various non-genotoxic hepatotoxins have been shown to induce
liver tumours in rodents (Drinkwater et al., 1990; Butterworth &
Goldsworthy, 1991; Grasso & Hinton, 1991). High doses and prolonged
exposure increase the frequency of mutations and the likelihood of
neoplastic transformation at the cellular level by still hypothetical
mechanisms, such as expression of protooncogenes and growth factors
(Thompson et al., 1986; Dubois, 1990). In rodents, the mechanism is
believed to be increased liver-cell proliferation due to repeated
hepatocellular necrosis-regeneration cycles, leading to the
development of foci of phenotypically altered hepatocytes (so-called
'preneoplastic lesions'), which finally progress to neoplasms (Pitot
et al., 1990; Butterworth & Goldsworthy, 1991; Grasso & Hinton,
1991).
The occurrence of foci of altered hepatocytes is an important
link in the cascade of hepatotoxicity-induced liver tumorigenesis.
Although the different types of preneoplastic lesions can readily be
detected by conventional haematoxylin and eosin staining of liver
tissue sections (Bannasch & Zerban, 1994), as was done in the studies
of the toxicity of flumequine, no such lesions were reported in
previously evaluated studies, including the 18-month study of
carcinogenicity in CD-1 mice (Sibinski et al., 1977b; Annex 1,
reference 111). In the 13-week study in CD-1 mice, clear-cell foci of
altered hepatocytes, which are one type of preneoplastic lesion, were
observed in one male at 400 mg/kg bw per day and in one male and one
female at 800 mg/kg bw per day (Stewart, 1995). The lesions were not
characterized histochemically.
In a special study to assess marker enzymes of hepatic
preneoplastic lesions, the effect of flumequine on the activity of
gamma-glutamyltransferase and glutathione S-transferase, which in
its placental form is a characteristic marker of foci of
phenotypically altered hepatocytes (Bannasch & Zerban, 1994), was
investigated in homogenates of livers from some of the CD-1 mice used
in the 13-week study. The activity of gamma-glutamyltransferase
remained unchanged. Administration of flumequine at doses of 400 and
800 mg/kg bw per day resulted in marked stimulation of glutathione
S-transferase activity towards 1-chloro-2,4-dinitrobenzene in
females, whereas the enzyme activity in males was only slightly
affected. Doses up to 100 mg/kg bw per day had no effect (Stewart,
1996). Because of its inadequate design, this study did not allow a
valid assessment of marker enzymes of preneo-plastic lesions, which
are confined to a small number of phenotypically altered hepatocytes.
Therefore, this study was not considered further in the evaluation.
In conclusion, flumequine was considered to be a non-genotoxic
hepatocarcinogen, and the induction of hepatocellular
necrosis-regeneration cycles by hepatotoxicity was considered to be
the relevant mechanism for induction of liver tumours. Since cytotoxic
effects are a prerequiste of hepatocarcinogenicity, tumours are
induced only at hepatotoxic doses (Cohen & Ellwein, 1990; Pitot
et al., 1990). Therefore, the NOEL for the hepatotoxicity of
flumequine, 25 mg/kg bw per day, was considered to be the threshold
for both the hepatotoxicity and the associated carcinogenicity of
flumequine. In evaluating the safety of flumequine, it must be kept in
mind that the NOEL for hepatoxic lesions was derived from a short-term
(13 weeks) study and was extrapolated to the level required for tumour
formation observed at the end of a lifetime study (18 months) in mice.
2.1.2 Special studies on human intestinal flora
No experimental data on the effect of flumequine on the bacteria
of the human gut microflora were available at the time of the previous
evaluation (Annex 1, reference 110). Additional studies have been
provided to assess the effects of flumequine and its 7-hydroxy
metabolite on bacterial isolates from human intestinal microflora
in vitro. Studies in vivo have not been performed.
In the first study, the minimum concentrations resulting in 50%
inhibition (MIC50) and 90% inhibition (MIC90) and the geometric
mean of the MIC50 were determined for 100 bacterial strains isolated
from the faeces of healthy volunteers, comprising 10 isolates of 10
aerobic and anaerobic bacterial genera typical of the human gut
microflora. The tests were performed in agar with serial dilutions
under anaerobic and aerobic (Escherichia coli) conditions. Three
bacterial tester strains were tested for reference. The inoculum
density was 107 colony forming units (cfu) per ml. The GLP status of
the study was not reported, but the protocol and conduct met accepted
standards for such studies. The results of the MIC determinations are
summarized in Table 1.
E. coli was the most sensitive species, the mean MIC50 value
for the 10 strains tested being 0.33 µg/ml. The mean MIC50 values
for the most sensitive predominant species isolated from the human
gastrointestinal tract, Clostridium and Fusobacterium, were 0.95
and 1.0 µg/ml, respectively (Richez, 1994a).
In a second set of experiments under similar experimental
conditions, the MIC values of 7-hydroxyflumequine were determined
against 10 strains each of E. coli, Clostridium spp., and
Fusobacterium spp. E. coli was much less sensitive to the
metabolite than to the parent substance, with no inhibition at a
concentration of 2 µg/ml 7-hydroxyflumequine and 100% inhibition at
4 µg/ml. The strains of Clostridium and Fusobacterium spp. were
not sensitive to the highest concentration tested (16 µg/ml) (Richez,
1995).
Table 1. Susceptibility of human intestinal bacteria to flumequine in vitro
Bacterial species MIC (µg/ml)
(10 strains each)
Range MIC50 MIC90 Geometric mean
E. coli (aerobic) 0.25-0.50 0.33 0.48 0.47
E. coli (anaerobic) 0.25-0.50 0.33 0.48 0.47
Streptococcus spp. 16- &gt 32 &gt 32 &gt 32 &gt 32
Proteus spp. &gt 32 &gt 32 &gt 32 &gt 32
Lactobacillus spp. &gt 32 &gt 32 &gt 32 &gt 32
Bifidobacterium spp. &gt 32 &gt 32 &gt 32 &gt 32
Bacteroides fragilis 16- &gt 32 &gt 32 &gt 32 &gt 32
Eubacterium spp. &gt 32 &gt 32 &gt 32 &gt 32
Clostridium spp. 0.50-2.0 0.95 1.70 1.32
Fusobacterium spp. 1.0-32 1.0 5.1 3.25
Peptostreptococcus spp. &gt 32 &gt 32 &gt 32 &gt 32
In a further experiment, the influence of inoculum size on the
MIC was investigated for 10 strains of E. coli isolated from human
faeces. Each strain was tested under anaerobic and aerobic conditions
with an inoculum of either 107 or 109 cfu/ml. No effect of inoculum
density was seen (Richez, 1994b).
In a study reported in the open literature, the effects of
gastrointestinal factors and pH on the MIC50 of flumequine were
studied with relevant bacterial species. The tests were performed by
the broth dilution technique in the presence of cooked meat or a
combination of meat and milk, at pH values of 3-7.5. The obligate
anaerobes remained insensitive to flumequine (MIC50 &gt 40 µg/ml),
whereas the MIC50 values for the E. coli strains were increased by
two- to eightfold (Nouws et al., 1994).
The intestinal bioavailability of flumequine to enteric bacteria
was tested by giving 830 mg 14C-flumequine orally to five healthy
volunteers. The levels of radiolabel were then monitored in plasma,
urine, and faeces for up to five days. A total of 84% (76-92%)
radiolabel was recovered in excreta, with 9% (5.7-13%) in faeces and
75% (70-81%) in urine. It was concluded that about 10% of a dose of
flumequine is available to the gut microflora (Riker Laboratories,
Inc., 1994).
No studies were submitted on the selection of intestinal
bacterial resistance or on the inhibitory effects on microorganisms
used in industrial processing of foodstuffs of animal origin.
It was concluded that E. coli is the most sensitive of the
relevant bacterial strains of human gut microflora tested in vitro.
The absence of an effect of inoculum size indicates that flumequine
has similar reactivity even at the high bacterial density in the human
colon. The metabolite 7-hydroxyflumequine has markedly less
antibacterial activity and can be considered to exert no relevant
adverse effects on the human intestinal microflora. About 10% of
ingested flumequine is available to the microflora in the human gut.
The thirty-eighth Committee concluded that the most relevant
parameter in vitro for assessing the risk to human intestinal flora
is the geometric mean MIC against the most sensitive intestinal
microorganism (Annex 1, reference 97). The MIC50 of flumequine for
E. coli of 0.33 µg/ml should thus be considered the concentration
that has no effect on human intestinal microflora and be used to
establish the ADI. E. coli, which is very sensitive to
fluoroquinolones in general, is, however, a minor component of the
gastrointestinal flora (Moore & Moore, 1995). It is therefore more
appropriate to consider the effects of flumequine on the most
sensitive obligate anaerobes, which are the bacterial species that
predominate in the human gut (Moore & Moore, 1995). The MIC50 values
for Clostridium and Fusobacterium spp., 0.95-1.0 µg/ml, were
considered to be the concentrations with no effect on human intestinal
microflora.
Flumequine is a fluoroquinolone and thus has a broad spectrum of
activity against aerobic gram-negative bacteria. In humans, this class
of antimicrobial agents is used clinically for selective elimination
of potential aerobic and facultative anaerobic pathogens from the
gastrointestinal tract while preserving the predominant anaerobic
bacterial gut flora. Furthermore the administration of therapeutic
oral doses of fluoroquinolones such as ciprofloxacin and norfloxacin
to humans has not been shown to alter the intestinal bacterial ecology
or to weaken the barrier effect. Anaerobic bacteria such as
Bifidobacterium, Bacteroides, Eubacterium, Fusobacterium, and
Peptostreptococcus spp., the main components of the flora in the
human gastrointestinal tract, are largely unaffected by these
compounds (Midtvedt, 1990; Nord, 1995). When assessing the effects of
flumequine on human gastrointestinal flora, it is important to
interpret the MIC50 values for selected intestinal bacterial species
in the context of the overall gut ecosystem. Since the obligate
anaerobic bacteria that are predominantly isolated from the
gastrointestinal tract are relatively insensitive to fluoroquinolones,
disturbance of the human gut ecosystem by residues of flumequine is
unlikely.
3. COMMENTS
The Committee considered additional information on the induction
of arthropathy in young dogs, the hepatotoxic and liver
enzyme-inducing effects of flumequine in mice, the possible mechanism
of the hepatocarcinogenicity of flumequine, and its effect on human
gut microflora. The studies were carried out according to appropriate
standards for study protocol and conduct.
In order to test the effects of flumequine on articular
cartilage, it was administered as tablets to groups of 10
three-month-old beagle dogs at doses of 0, 15, 30, 60, or 150 mg/kg
bw per day for 13 weeks; four animals from each group were killed
after three weeks. The animals showed no overt clinical signs of
arthropathy. Gross necropsy revealed erosions of the joint surfaces in
two of 10 dogs at the highest dose and in one of 10 animals at the
lowest dose. Histopathological lesions of the articular cartilage were
observed in one of 10 dogs given 60 mg/kg bw per day and three of 10
dogs given 150 mg/kg bw per day. The severity of the lesions was
similar at three and 13 weeks. The Committee considered that the gross
lesions in the one animal at the lowest dose were not
compound-related, since no histopathological alterations were found
and no gross lesions were observed at the next two higher doses.
Therefore, the NOEL for induction of arthropathy in young dogs was 30
mg/kg bw per day.
In a 13-week study designed to investigate hepatotoxic lesions
and the activities of hepatic drug-metabolizing enzymes, flumequine
was administered to male CD-1 mice in the feed at doses equal to 0,
25, 50, 100, 400, or 800 mg/kg bw per day and to females at 0, 100,
400, or 800 mg/kg bw per day. The effects observed were reduced body
weight, significantly increased plasma activities of alanine and
aspartate aminotransferases, alkaline phosphatase and lactic
dehydrogenase, and increased liver weights at 400 and 800 mg/kg bw per
day. Histopathological examination of the liver revealed
dose-dependent hypertrophy, degenerative alterations, and
centrilobular hepatocellular necrosis. The hepatotoxic lesions were
more pronounced in male than in female mice and were observed at all
doses greater than 25 mg/kg bw per day. Increased mitosis was observed
only in males at the highest dose. Flumequine caused little or no
induction of hepatic cytochrome P450-dependent drug-metabolizing
enzymes or glucuronyltransferase when given at doses up to 800 mg/kg
bw per day. The NOEL was 25 mg/kg bw per day on the basis of induction
of hepatotoxic lesions in male mice.
At its forty-second meeting, the Committee noted that there was
evidence of compound-related tumorigenic effects in the livers of CD-1
mice. The hepatotumorigenic activity of flumequine was more pronounced
in male mice, which are known to be sensitive to liver tumour
induction. As the compound was inactive in a range of tests for
genotoxicity, including assays for gene mutation in bacteria and
mammalian cells in vitro and for chromosomal aberrations in
mammalian cells in vivo, the mechanism of this tumorigenesis was
unclear.
The present Committee noted that, although an inhibitory effect
of flumequine on mammalian topoisomerase II, leading to DNA damage,
was not investigated, bibliographical data on structurally related
fluoroquinolones indicate that this mechanism is unlikely to operate.
The Committee concluded that there is no evidence that flumequine has
genotoxic potential.
Non-genotoxic tumorigenesis in the liver can be due to various
mechanisms, including compound-related hormonal activity, peroxisomal
proliferation, induction of hepatic drug-metabolizing enzymes, and
hepatotoxicity.
The toxicological database, including studies of reproductive
toxicity, revealed no evidence for any hormonal activity of
flumequine. In short- and long-term studies of toxicity in mice, no
histopathological alterations of liver cells that indicate peroxisomal
proliferation were reported. Induction of the hepatic cytochrome P450
enzyme system can be excluded by the results of the 13-week study in
CD-1 mice.
Flumequine is hepatotoxic, causing hepatocellular degeneration
and focal necrosis in male and female mice, which was followed by a
mitogenic response in male mice at the highest dose in the 13-week
study described above. The dose-related severity of these hepatotoxic
lesions parallelled the incidence of benign and malignant liver
tumours. Various non-genotoxic hepatotoxins have been shown to induce
liver tumours. The mechanism is believed to be increased liver-cell
proliferation due to repeated hepatocellular necrosis-regeneration
cycles, leading to the development of foci of phenotypically altered
hepatocytes (so-called 'preneoplastic lesions'), which finally
progress to neoplasms. In the 13-week study in CD-1 mice, clear-cell
foci of altered hepatocytes, which are one type of preneoplastic
lesion, were observed in one male at 400 mg/kg bw per day and in one
male and one female at 800 mg/kg bw per day.
In a study to assess marker enzymes of hepatic preneoplastic
lesions, the effects of flumequine on the activity of
gamma-glutamyltransferase and glutathione S-transferase were
investigated in homogenates of livers from some of the mice used in
the 13-week study. Because of its inadequate design, including the
lack of histochemical characterization of the foci of altered
hepatocytes, this study was not considered further in the evaluation.
The Committee considered that induction of hepatocellular
necrosis-regeneration cycles by hepatotoxicity is the relevant
mechanism for induction of liver tumours by flumequine. Therefore, the
NOEL for the hepatotoxicity of flumequine, 25 mg/kg of bw per day, was
considered to be the threshold for both the hepatotoxicity of
flumequine and its associated carcinogenicity. The Committee noted
that hepatotoxicity would have been better explored in a study of
longer duration.
The effect of flumequine on human intestinal microflora was
assessed by determining the MIC50 values for 100 bacterial strains
isolated from human faeces, comprising 10 isolates from 10 aerobic and
anaerobic bacterial genera typical of the human gut microflora. These
included Escherichia coli, Streptococcus spp., Proteus spp.,
Lactobacillus spp., Bifidobacterium spp., Bacteroides fragilis, Eubacterium spp., Clostridium spp., Fusobacterium spp., and
Peptostreptococcus spp. The inoculum density was 107 colony
forming units per ml. E. coli was the most sensitive bacterial
species tested, with an MIC50 value of 0.33 µg/ml. The MIC50 value
was not dependent on the size of the inoculum. E. coli was markedly
less sensitive to 7-hydroxyflumequine, with an MIC50 value of 4
µg/ml. The mean MIC50 values for the most sensitive predominant
species typically isolated from the human gastrointestinal tract,
Fusobacterium and Clostridium, were 1.0 and 0.95 µg/ml,
respectively. In a study of the influence of gastrointestinal factors
and pH on the MIC50 values of flumequine for relevant bacterial
species of the human gastrointestinal tract, the values for obligate
anaerobes were unaffected, whereas the MIC50 values for E. coli
strains were increased by two- to eightfold.
The upper limit of the ADI based on the antimicrobial activity of
flumequine on human gut flora was calculated on the basis of the
formula described on p. 12 as follows:
Upper limit 1 µg/ga × 220 g
of ADI =
0.1b × 1c × 60 kg
= 37 µg/kg bw
a Mean MIC50 for the most sensitive predominant bacterial
species, Fusobacterium and Clostridiumb Fraction of oral dose available to act on microorganisms in the
colon, based on a study in which 830 mg 14C-flumequine were
given orally to five healthy volunteers. The levels of radiolabel
were then monitored in plasma, urine, and faeces for up to five
days. A total of 84% (78-92%) of the radiolabel was recovered in
the excreta, with 9% (5.7-13%) in faeces and 75% (70-81%) in
urine. The Committee concluded that approximately 10% of
flumequine is available to the gut microflora.
c A safety factor of 1 was used because relevant and sufficient
microbiological data were provided.
4. EVALUATION
The Committee noted that flumequine belongs to the group of
antimicrobial fluoroquinolones that are active against aerobic
gram-negative bacteria. In humans, this class of antimicrobial agents
is used clinically for selective elimination of potential aerobic and
facultative anaerobic pathogens from the gastrointestinal tract while
preserving the predominant anaerobic bacterial gut flora. The
Committee also recognized that administration of therapeutic oral
doses of fluoroquinolones such as ciprofloxacin and norfloxacin to
humans has no appreciable effect on the intestinal bacterial ecology
or on the barrier effect. In addition, anaerobic bacteria such as
Bifidobacterium, Bacteroides, Eubacterium, Fusobacterium, and
Peptostreptococcus spp., the main components of the human gut flora,
are largely unaffected by these compounds. E. coli, however, which
is very sensitive to fluoroquinolones in general, is a minor component
of the gastrointestinal flora. The Committee considered that, in
assessing the effects of flumequine on the bacteria of the human
gastrointestinal flora, the MIC50 values for the selected intestinal
bacterial species should be interpreted in the context of the overall
ecosystem of the gastrointestinal tract. Since the obligate anaerobic
bacteria that predominate in the gastrointestinal tract are relatively
insensitive to fluoroquinolones, disturbance of the human gut
ecosystem by residues of flumequine is unlikely. Therefore the
Committee decided to base the ADI on the toxicological properties of
flumequine and not on its effect on the intestinal microflora.
The Committee considered the NOEL of 25 mg/kg bw per day for
hepatotoxicity in male CD-1 mice in the 13-week study to be the most
appropriate toxicological end-point for consumer safety. An ADI of
0-30 µg/kg bw was established by applying a 1000-fold safety factor,
which was chosen to account for the short duration of the study and
the lack of histochemical characterization of the foci of altered
hepatocytes.
5. REFERENCES
Bannasch, P. & Zerban, H. (1994) Preneoplastic and neoplastic lesions
of the rat liver. In: Bannasch, P & Gśssner, W., eds, Pathology of
Neoplasia and Preneoplasia in Rodents, Stuttgart, Schattauer, pp.
18-30.
Brown, S.A. (1996) Fluoroquinolones in animal health. J. Vet.
Pharmacol. Ther., 19, 1-14.
Butterworth, B.E. & Goldsworthy, T.L. (1991) The role of cell
proliferation in multistage carcinogenesis. Proc. Soc. Exp. Biol.
Med., 198, 683-687.
Cohen, S.M. & Ellwein, L.B. (1990) Cell proliferation in
carcinogenesis. Science, 249, 1007-1011.
Diwan, B.A., Rice, J.M. & Ward, J.M. (1990) Strain-dependent effects
of phenobarbital on liver tumour promotion in inbred mice. In: Mouse
Liver Carcinogenesis: Mechanisms and Species Comparisons, New York,
Alan R. Liss, Inc., pp. 69-83.
Drinkwater, N.R., Hanigan, M.H. & Kemp, C.J. (1990) Genetic and
epigenetic promotion of murine hepatocarcinogenesis. In: Mouse Liver
Carcinogenesis: Mechanisms and Species Comparisons, New York, Alan R.
Liss, Inc., pp. 163-176
Dubois, R.N. (1990) Early changes in gene expression during liver
regeneration. What do they mean? Hepatology, 11, 1079-1082.
Faccini, J.M., Butler, W.R., Friedman, J.C., Hess, R., Reznik, G.K.,
Ito, N., Hayashi, Y. & Williams, G.M. (1992) IFSTP guidlines for the
design and interpretation of the chronic rodent carcinogenicity
bioassay. Exp. Toxicol. Pathol., 44, 443-456.
Gough, A., Barsoum, N., Mitchell, L., McGuire, E. & de la Iglesias, F.
(1979) Juvenile canine drug-induced arthropathy: Clinicopathological
studies on articular lesions caused by oxolinic acid and pipemidic
acid. Toxicol. Appl. Pharmacol., 51, 177-187.
Grasso, R. & Hinton, R.H. (1991) Evidence for and possible mechanisms
of non-genotoxic carcinogenicity in rodent liver. Mutat. Res., 248,
271-290
Huff, A.C. & Kreuzer, K.N. (1991) The mechanism of antitumour drug
action in a simple bacteriophage model system. In: Potmesil, M. &
Kohn, K., eds, DNA Topoisomerases in Cancer [publishers not given],
pp. 215-229. Submitted to WHO by Sanofi Santé Nutrition Animale,
Libourne, France.
Hussy, P., Maass, G., Tummler, B., Grosse, F. & Schomburg, U. (1986)
Effect of 4-quinolones and novobiocin on calf thymus DNA polymerase a
primase complex, topoisomerase I and II, and growth of mammalian
lymphoblasts. Antimicrob. Agents Chemother., 29, 1073-1078.
Liu, L.F. & Wang, J.C. (1991) Biochemistry of DNA topoisomerases and
their poisons. In: Potmesil, M. & Kohn, K., eds, DNA Topoisomerases in
Cancer [publishers not given], pp. 13-22. Submitted to WHO by Sanofi
Santé Nutrition Animale, Libourne, France.
Marzin, D. (1996) Flumequine MRLs file. Unpublished report from
Laboratoire de Toxicologie, Insitut Pasteur de Lille, France.
Submitted to WHO by Sanofi Santé Nutrition Animale, Libourne, France.
McClain, R.M. (1990) Mouse liver tumours and microsomal
enzyme-inducing drugs: Experimental and clinical perspectives with
phenobarbital. In: Mouse Liver Carcinogenesis: Mechanisms and Species
Comparisons, New York, Alan R. Liss, Inc., pp. 345-365.
Midtvedt, T. (1990) The influence of quinolones on the faecal flora.
Scand. J. Infect. Dis., 68 (Suppl.), 14-18.
Moore, W.E.C. & Moore, L.H. (1995) Intestinal floras of populations
that have high risk of colon cancer. Appl. Environ. Microbiol., 61,
3202-3207.
Nelson, R.A., Case, M.T., Glick, P.R. & Steffen, G.R. (1972) Ninety
(90) day oral subacute toxicity of flumequine in beagle dogs.
Unpublished report from Riker Laboratories Inc., St Paul, MN, USA.
Submitted to WHO by Sanofi Santé Nutrition Animale, Libourne, France.
Nord, C.E. (1995) Effect of quinolones on the human intestinal
microflora. Drugs, 49 (Suppl.), 81-85.
Norrby, S.R. & Lietman, P.S. (1993) Safety and tolerability of
fluoroquinolones. Drugs, 45 (Suppl. 3), 59-64.
Nouws, J.F.M., Kuiper, H., van Klingeren, B. & Kruyswijk, P.G. (1994)
Establishment of a microbiologically acceptable daily intake of
antimicrobial drug residue. Vet. Q., 16, 152-156.
Pitot, H.C., Dragan, Y., Xu, Y., Pyron, M., Laufer, C. & Rizvi, T.
(1990) Role of altered hepatocyte foci in the stages of
carcinogenesis. In: Mutation and the Environment, New York, Wiley-
Liss, Inc., pp. 81-95. Submitted to WHO by Sanofi Santé Nutrition
Animale, Libourne, France.
Purchase, I.F.H. (1994) Current knowledge of mechanisms of
carcinogenicity: Genotoxins versus non-genotoxins. Hum. Exp. Toxicol.,
13, 17-28.
Richez, P. (1994a) Antibacterial activity of flumequine against human
gut microflora. Unpublished report No. SF008 from DataVet, Vendargues,
France. Submitted to WHO by Sanofi Santé Nutrition Animale, Libourne,
France.
Richez, P. (1994b) Antibacterial activity of flumequine against
Escherichia coli strains isolated from the human gut microflora: The
effect of the inoculum size. Unpublished report No. SF008-2 from
DataVet, Vendargues, France. Submitted to WHO by Sanofi Santé
Nutrition Animale, Libourne, France.
Richez, P. (1995) Antibacterial activity of 7-OH flumequine against
human gut microflora. Unpublished report No. SF008-3 from DataVet,
Vendargues, France. Submitted to WHO by Sanofi Santé Nutrition
Animale, Libourne, France.
Riker Laboratories, Inc. (1994) Metabolism of R802 (flumequine) in
humans. Unpublished report form Riker Laboratories Inc., St Paul, MN,
USA. Submitted to WHO by Sanofi Santé Nutrition Animale, Libourne,
France.
Sato, K., Hoshing, K., Une, T. & Osada, Y. (1989) Inhibitory effects
of ofloxacin on DNA gyrase of Escherichia coli and topoisomerase II of
bovine calf thymus. Rev. Infect. Dis., 2 (Suppl. 5), 5915-5916.
Shaw, I.C. & Jones, H.B. (1994) Mechanisms of non-genotoxic
carcinogenesis. TiPS, 15, 89-93.
Sibinski, L.J., Steffen, G.R. & Case, M.T. (1977a) Two-year oral
toxicity-carcinogenicity study of R-802 (flumequine) in rats.
Unpublished report from Riker Laboratories, Inc., St Paul, MN, USA.
Submitted to WHO by Sanofi Santé Nutrition Animale, Libourne, France.
Sibinski, L.J., Steffen, G.R. & Case, M.T. (1977b) Eighteen (18) month
oral carcinogenicity study of R-802 (flumequine) in mice. Unpublished
report from Riker Laboratories, Inc., St Paul, MN, USA. Submitted to
WHO by Sanofi Santé Nutrition Animale, Libourne, France.
Sibinski, L.J., Steffen, G.R. & Case, M.T. (1979) Special 18-month
toxicity study of R-802 (flumequine) in male mice. Unpublished report
from Riker Laboratories, Inc., St Paul, MN, USA. Submitted to WHO by
Sanofi Santé Nutrition Animale, Libourne, France.
Stewart, J.S. (1995) Flumequine: Toxicity study by dietary
administration to CD-1 mice for 13 weeks. Unpublished report No.
94/0678 from Pharmaco LSR, Eye, Suffolk, United Kingdom. Submitted to
WHO by Sanofi Santé Nutrition Animale, Libourne, France.
Stewart, J.S. (1996) Flumequine: Supplementary analysis to a toxicity
study by dietary administration to CD-1 mice for 13 weeks. Unpublished
report No. 96/0443 from Huntingdon Life Sciences Ltd, Eye, Suffolk,
United Kingdom. Submitted to WHO by Sanofi Santé Nutrition Animale,
Libourne, France.
Thompson, N.L., Mead, J.E., Braun, L., Goyette, M., Shank, P.R. &
Fausto, N. (1986) Sequential protooncogene expression during rat liver
regeneration. Cancer Res., 46, 3111-3117.
Woehrle, F. (1996) Flumiquil comprimés 200 mg -- 13 week oral (tablet)
tolerance study in the young beagle dog with interim necropsy after 3
weeks. Study No. 311/536. Unpublished report from Pharmakon Europe,
L'Arbresle, France. Submitted to WHO by Sanofi Santé Nutrition
Animale, Libourne, France.